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n-Butylidenephthalide induced apoptosis in the A549 human lung adenocarcinoma cell line by coupled down-regulation of AP-2alpha and telomerase activity.

Wei CW, Lin CC, Yu YL, Lin CY, Lin PC, Wu MT, Chen CJ, Chang W, Lin SZ, Chen YL, Harn HJ - Acta Pharmacol. Sin. (2009)

Bottom Line: Xenograft mice were used as a model system to study the cytotoxic effect of n-BP in vivo.We saw an inhibition of tumor growth when nude mice carrying A549 subcutaneous xenograft tumors were treated with n-BP.Immunohistochemistry of this tumor tissue also showed a decrease in the expression of hTERT.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biomedical Nutrition, College of Medicines and Nursings, Hungkuang University, Taichung, Taiwan, China;

ABSTRACT

Aim: To investigate the role of hTERT gene expression and AP-2alpha in n-butylidenephthalide (n-BP)-induced apoptosis in A549 lung cancer cells.

Methods: Viability of A549 cells was measured by MTT assay. Protein expression was determined by Western blot. Telomerase activity was measured using the modified telomere repeat amplification protocol (TRAP) assay. Xenograft mice were used as a model system to study the cytotoxic effect of n-BP in vivo. The morphology of tumor was examined by immunohistochemical staining.

Results: The growth of A549 lung cancer cells treated with n-BP was significantly inhibited. Telomerase activity and hTERT mRNA expression were determined by telomeric repeat amplification protocol and reverse transcription-polymerase chain reaction, respectively. n-BP inhibited telomerase activity and hTERT mRNA expression in A549 cells while overexpression of hTERT could abolish BP-induced growth inhibition in the A549 cells. We also showed that hTERT promoter activity in the presence of n-BP was mediated via AP-2alpha. We saw an inhibition of tumor growth when nude mice carrying A549 subcutaneous xenograft tumors were treated with n-BP. Immunohistochemistry of this tumor tissue also showed a decrease in the expression of hTERT.

Conclusion: The antiproliferative effects of n-BP on A549 cells in vitro and in vivo suggest a novel clinical application of this compound in the treatment of lung cancers.

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Inhibition of ERK expression and enhanced growth inhibition by MEK inhibitor PD98059. A, MTT assay of A549 cells with culture or serum-containing medium pretreated with the MEK1/2 inhibitor PD98059 (12.5, 25, and 50 μmol/L), the PKC inhibitor GF109203X (5, 10, and 20 μmol/L), or the PI3K/AKT inhibitor LY294002 (5, 10, and 20 μmol/L) for 1 h and then treated with 50 μg/mL BP for 24 and 48 h. Lane 1 shows A549 cells treated with serum containing media and no test compound as a negative control. The data represent the means±SD of three different experiments. bP<0.05, cP<0.01 vs the vehicle. B, Western blot analysis of ERK, phosphor-ERK (pERK), PKC, phosphor-PKC (pPKC), AKT, phosphor-AKT (pAKT), GSK-3β, and phosphor-GSK-3β (pGSK-3β) in A549 cells after treatment with 50 μg/mL BP for the indicated times. The expression of β-actin was used as an internal control. (C) Inhibition of AP-2 and enhancement of cleaved caspase-3 expression by MEK inhibitor in the BP-induced growth inhibition. A549 cells were in incubated in the presence or absence of the MEK inhibitor PD98059 for 1 h and then treated with BP for 12 h. Western blot analysis was performed for AP-2α, cleaved caspase-3, and expression of β-actin was used as an internal control.
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fig5: Inhibition of ERK expression and enhanced growth inhibition by MEK inhibitor PD98059. A, MTT assay of A549 cells with culture or serum-containing medium pretreated with the MEK1/2 inhibitor PD98059 (12.5, 25, and 50 μmol/L), the PKC inhibitor GF109203X (5, 10, and 20 μmol/L), or the PI3K/AKT inhibitor LY294002 (5, 10, and 20 μmol/L) for 1 h and then treated with 50 μg/mL BP for 24 and 48 h. Lane 1 shows A549 cells treated with serum containing media and no test compound as a negative control. The data represent the means±SD of three different experiments. bP<0.05, cP<0.01 vs the vehicle. B, Western blot analysis of ERK, phosphor-ERK (pERK), PKC, phosphor-PKC (pPKC), AKT, phosphor-AKT (pAKT), GSK-3β, and phosphor-GSK-3β (pGSK-3β) in A549 cells after treatment with 50 μg/mL BP for the indicated times. The expression of β-actin was used as an internal control. (C) Inhibition of AP-2 and enhancement of cleaved caspase-3 expression by MEK inhibitor in the BP-induced growth inhibition. A549 cells were in incubated in the presence or absence of the MEK inhibitor PD98059 for 1 h and then treated with BP for 12 h. Western blot analysis was performed for AP-2α, cleaved caspase-3, and expression of β-actin was used as an internal control.

Mentions: To determine whether MAPK/ERK, PKC, or PI3K/AKT/GSK3β play a role in BP-induced growth inhibition of A549 cells, these cells were treated with BP in the presence or absence of the mitogen-activated protein kinase kinase 1/2 inhibitor PD98059 (12.5–50 μmol/L), PI3K/AKT/GSK3β inhibitor LY294002 (5–20 μmol/L), or the PKC inhibitor GF1023X (5–20 μmol/L). It was shown that all the three inhibitors enhanced BP-induced growth inhibition in a concentration-dependent manner (Figure 5A). To determine which above mentioned pathways were involved in BP-induced growth inhibition, the effect of BP on ERK inhibition was assessed. Inhibition of phosphor-ERK protein expression was observed after exposure of A549 cells to BP. PKC protein expression had no obvious changes whereas phosphor-AKT protein expression was activated after drug treatment (Figure 5B). To investigate a possible role for ERK in the regulation of AP-2α, A549 cells were treated with BP in the presence or absence of the MAPK inhibitor PD98059. Using Western blot analysis, PD98059 had synergistic effects with n-BP in suppressing AP-2α and enhancing cleaved caspase-3 protein levels (Figure 5C). These observations suggest that inhibition of the ERK1/2 signaling pathway was involved in n-BP-inhibited AP-2α expression and led to apoptosis in A549 cells.


n-Butylidenephthalide induced apoptosis in the A549 human lung adenocarcinoma cell line by coupled down-regulation of AP-2alpha and telomerase activity.

Wei CW, Lin CC, Yu YL, Lin CY, Lin PC, Wu MT, Chen CJ, Chang W, Lin SZ, Chen YL, Harn HJ - Acta Pharmacol. Sin. (2009)

Inhibition of ERK expression and enhanced growth inhibition by MEK inhibitor PD98059. A, MTT assay of A549 cells with culture or serum-containing medium pretreated with the MEK1/2 inhibitor PD98059 (12.5, 25, and 50 μmol/L), the PKC inhibitor GF109203X (5, 10, and 20 μmol/L), or the PI3K/AKT inhibitor LY294002 (5, 10, and 20 μmol/L) for 1 h and then treated with 50 μg/mL BP for 24 and 48 h. Lane 1 shows A549 cells treated with serum containing media and no test compound as a negative control. The data represent the means±SD of three different experiments. bP<0.05, cP<0.01 vs the vehicle. B, Western blot analysis of ERK, phosphor-ERK (pERK), PKC, phosphor-PKC (pPKC), AKT, phosphor-AKT (pAKT), GSK-3β, and phosphor-GSK-3β (pGSK-3β) in A549 cells after treatment with 50 μg/mL BP for the indicated times. The expression of β-actin was used as an internal control. (C) Inhibition of AP-2 and enhancement of cleaved caspase-3 expression by MEK inhibitor in the BP-induced growth inhibition. A549 cells were in incubated in the presence or absence of the MEK inhibitor PD98059 for 1 h and then treated with BP for 12 h. Western blot analysis was performed for AP-2α, cleaved caspase-3, and expression of β-actin was used as an internal control.
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Related In: Results  -  Collection

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fig5: Inhibition of ERK expression and enhanced growth inhibition by MEK inhibitor PD98059. A, MTT assay of A549 cells with culture or serum-containing medium pretreated with the MEK1/2 inhibitor PD98059 (12.5, 25, and 50 μmol/L), the PKC inhibitor GF109203X (5, 10, and 20 μmol/L), or the PI3K/AKT inhibitor LY294002 (5, 10, and 20 μmol/L) for 1 h and then treated with 50 μg/mL BP for 24 and 48 h. Lane 1 shows A549 cells treated with serum containing media and no test compound as a negative control. The data represent the means±SD of three different experiments. bP<0.05, cP<0.01 vs the vehicle. B, Western blot analysis of ERK, phosphor-ERK (pERK), PKC, phosphor-PKC (pPKC), AKT, phosphor-AKT (pAKT), GSK-3β, and phosphor-GSK-3β (pGSK-3β) in A549 cells after treatment with 50 μg/mL BP for the indicated times. The expression of β-actin was used as an internal control. (C) Inhibition of AP-2 and enhancement of cleaved caspase-3 expression by MEK inhibitor in the BP-induced growth inhibition. A549 cells were in incubated in the presence or absence of the MEK inhibitor PD98059 for 1 h and then treated with BP for 12 h. Western blot analysis was performed for AP-2α, cleaved caspase-3, and expression of β-actin was used as an internal control.
Mentions: To determine whether MAPK/ERK, PKC, or PI3K/AKT/GSK3β play a role in BP-induced growth inhibition of A549 cells, these cells were treated with BP in the presence or absence of the mitogen-activated protein kinase kinase 1/2 inhibitor PD98059 (12.5–50 μmol/L), PI3K/AKT/GSK3β inhibitor LY294002 (5–20 μmol/L), or the PKC inhibitor GF1023X (5–20 μmol/L). It was shown that all the three inhibitors enhanced BP-induced growth inhibition in a concentration-dependent manner (Figure 5A). To determine which above mentioned pathways were involved in BP-induced growth inhibition, the effect of BP on ERK inhibition was assessed. Inhibition of phosphor-ERK protein expression was observed after exposure of A549 cells to BP. PKC protein expression had no obvious changes whereas phosphor-AKT protein expression was activated after drug treatment (Figure 5B). To investigate a possible role for ERK in the regulation of AP-2α, A549 cells were treated with BP in the presence or absence of the MAPK inhibitor PD98059. Using Western blot analysis, PD98059 had synergistic effects with n-BP in suppressing AP-2α and enhancing cleaved caspase-3 protein levels (Figure 5C). These observations suggest that inhibition of the ERK1/2 signaling pathway was involved in n-BP-inhibited AP-2α expression and led to apoptosis in A549 cells.

Bottom Line: Xenograft mice were used as a model system to study the cytotoxic effect of n-BP in vivo.We saw an inhibition of tumor growth when nude mice carrying A549 subcutaneous xenograft tumors were treated with n-BP.Immunohistochemistry of this tumor tissue also showed a decrease in the expression of hTERT.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biomedical Nutrition, College of Medicines and Nursings, Hungkuang University, Taichung, Taiwan, China;

ABSTRACT

Aim: To investigate the role of hTERT gene expression and AP-2alpha in n-butylidenephthalide (n-BP)-induced apoptosis in A549 lung cancer cells.

Methods: Viability of A549 cells was measured by MTT assay. Protein expression was determined by Western blot. Telomerase activity was measured using the modified telomere repeat amplification protocol (TRAP) assay. Xenograft mice were used as a model system to study the cytotoxic effect of n-BP in vivo. The morphology of tumor was examined by immunohistochemical staining.

Results: The growth of A549 lung cancer cells treated with n-BP was significantly inhibited. Telomerase activity and hTERT mRNA expression were determined by telomeric repeat amplification protocol and reverse transcription-polymerase chain reaction, respectively. n-BP inhibited telomerase activity and hTERT mRNA expression in A549 cells while overexpression of hTERT could abolish BP-induced growth inhibition in the A549 cells. We also showed that hTERT promoter activity in the presence of n-BP was mediated via AP-2alpha. We saw an inhibition of tumor growth when nude mice carrying A549 subcutaneous xenograft tumors were treated with n-BP. Immunohistochemistry of this tumor tissue also showed a decrease in the expression of hTERT.

Conclusion: The antiproliferative effects of n-BP on A549 cells in vitro and in vivo suggest a novel clinical application of this compound in the treatment of lung cancers.

Show MeSH
Related in: MedlinePlus